| In the past few decades, the incidence of obesity and its associated metabolic disorders, including non-alcoholic fatty liver disease (NAFLD), hyperlipidemia and type 2 diabetes escalating, which challenge the modern global health care. NAFLD is the now most common chronic liver disease, its prevalence in obese and diabetic population was significantly higher than the normal. In addition, these diseases can further lead to atherosclerosis, coronary heart disease (CHD) and other cardiovascular diseases. Currently lifestyle modifications are difficult to maintain weight loss and thiazolidinediones pose safety issues and weght-gain. Therefore, focus on the development of traditional Chinese medicine to alleviate lipid disorders and hepatic steatosis is very necessary. The most significant pathological changes of NAFLD are hepatic lipids, especially triglyceride (TG) abnormal deposited in hepatocyte, which results in lipotoxicity and hepatic dysfunction. The specific mechanisms of ectopic hepatic lipid deposition are not well understood, but the interaction of lipogenesis (fatty acid de novo synthesis), lipid delivery and transport (import or export) and FFA oxidation is certaintly involved. Therapies capable of regulating of these processes or metabolic enzymes may have a role on the treatment of NAFLD. At present, for NAFLD clinically relevant drugs for NAFLD such as thiazolidinediones, TZDs and lifestyle changes, which are not satisfactory in the efficacy and safety issues. Therefore, take advantage of our focus on the development of traditional Chinese medicine to improve blood lipid disorders and hepatic steatosis protection becomes necessary.Prof. Li proposed Tangshen formula (TSF) based on predecessors experience and clinical experience in order to make the treatment of diabetic nephropathy (DN). Multi-center randomized controlled trial study showed which can effectively reduce the 24 h urine protein and increase glomerular filtration rate; but whether it is effective for lipid metabolism associated with diabetes and the specific molecular mechanisms? In our study, we investigated the role of Tangshen formular (TSF) on the modulation of dyslipidemia, hepatic steatosis and its possible mechanisms invovled several metabolic pathways of lipid metaboliam using db/db mice, a useful animal model of NAFLD. Mainly focus on 1) AMPK/SREBPs mediated lipogenesis pathway; 2) AMPK/ Sirtl/PGCla mediated fatty acid β oxidation; 3) PI3K/Akt/mTOR signaling pathway and the gluconeogenesis; 4) The liver and white adipose tissue macrophage activation associateed to lipid metabolism regulation.Aims:Use of spontaneous obese and type 2 diabetes db/db mice to observe the effect of TSF on the regulation of lipid metabolism and the role of protecting the hepatic steatosis and its potential mechanisms invovled hepatic lipid accumulation.Methods:Eight weeks old db/db and db/m mice were randomly divided into three subgroups, (db/m, db/db, db/db+TSF), and all mice were fed with a normal diet. After two weeks adaptation, mice were maintained on TSF treatment for 12 weeks and were then sacrificed under anesthesia after overnight fast. Body weight was recorded weekly. Animal blood and tissues samples (including liver, skeletal muscle and white adipose tissue) were collected for further analyses.The liver washed with cold PBS solution, dry it out and weight; one part of the liver tissue stored in liquid nitrogen, another deposited-80℃ directly for subsequent frozen sections; the rest of the liver fixed into the neutral formalin for pathological analysis. Skeletal muscle and white adipose tissue immediately stored in liquid nitrogen for further analyses.Chromatographic analysis of TSF was used by HPLC fingerprint spectrum. H&E and oil red O staining investigated the hepatic steatosis. The hepatic expression of pAMPK/SREBPs and its target genes involved in lipogenesis were assessed by Western blot, real-time PCR, IHC and confocal microscopy. We also assessed metabolic enzymes associated with fatty acid β oxidation in liver and skeletal muscle, impact of using the above method for detecting Tangshen side of each group of mice liver tissue The expression of PI3K/Akt/mTOR and glycogen synthesis were determined by western blot and PAS staining. Markers of hepatic and white adipose tissue macrophage activation were assessed by IHC and Real-time PCR assays.Results:1. Effects of TSF on lipid metabolism and hepatic steatosis in db/db mice. TSF diminished weight gain and liver index of db/db mice significantly after 12 weeks chronic treatment. At 22 weeks old age it was apparent that the db/db mice increased the size and demonstrated fatty liver and that TSF prevented these changes. TSF could significantly reduced serum ALT, AST, TG, TC, LDL-C, LDL-C/HDL-C and FFA, improve lipid profile and hepatic function, but does not affect FBG. Histological examination of liver sections by H&E and oil red O staining revealed a loss of cellular integrity and the deposition of large lipid droplets in the db/db mice livers but not the db/db+TSF group.2. The potential mechanisms of TSF alleviated hepatic steatosis in db/db mice2.1 Effects of TSF on DNL of fatty acid and its downstream target molecules. We found that TSF stimulated AMPK phosphorylation and inhibited nuclear translocation of SREBP1 in the liver of db/db mice. In addition, our results showed that TSF inhibited the protein and genes expression of hepatic lipogenic enzymes in the db/db mice, such as FAS, SCD1, HMGCR and HMGCS by IHC and western blot. We also found protein expression of phosphorylation of ACC, an AMPK downstream target, and the mRNA levels of AdipoRl/2 were decreased and restored by TSF.2.2 Effects of TSF on lipid oxidation in db/db mice liver and skeletal muscle. We found that TSF significantly increased the expression of Sirtl, PGC1α, PPARα, MLYCD and CPT1A in the liver of db/db mice. TSF also increased the expression of genes involved in fatty acid oxidation such as Acoxl and Acadm, and decreased the expression of LXRa and LOX1. In skeletal muscle of db/db mice, TSF increased the protein levels of pAMPK, Sirtl, PGCla and MLYCD. Moreover, we found that TSF therapy prominently upregulated CPT1B and Acadm genes expression while downregulated Ucp2/3. Notably, we detected a marked downregulation of both LPL and CD36 in db/db mice subjected to TSF administration, In addition, we found that TSF increased the expression of genes that promote mitochondrial biogenesis in skeletal muscle.2.3 Effects of TSF therapy on PI3K/Akt/mTOR cascade and macrophages activation. We found that TSF therapy markedly increased the protein levels of PI3K/Akt/and decreased the phosphorylation of mTOR in the liver of db/db mice. We also detected that TSF reduced the expression of genes involved in hepatic gluconeogenesis such as Pckl, G6pc. Glucokinase (GK), which catalyzes the first step of both glycogen and glycolysis, was found increased by TSF. We next study the effects of TSF on macrophsges in the liver and white adipose tissue; orally administrated TSF reduced the expression levels of macrophage markers such as CD68 and F4/80. Interestingly, TSF reduced the expression of genes involvled in energy disipation such as Ucp2, and genes involved inflammatin such as Ccl2 in white adipose tissue. Notably, TSF reduced the levels of markers for classically activated M1 such as TNFa-but not the levels of markers for alternatively activated M2 such as Mrc1 and Arg1.Conclusions:TSF decreased the body weight, liver index, serum enzymes and lipid levels, which leads to amelioration of dyslipidemia and hepatic steatosis in db/db mice. Its effects may results from modulation of AMPK/SREBPs and target genes to inhibiting lipogenesis, augmenting fatty acid oxidation by Sirt/PGC1α, as well as regulating the PI3K/Akt /mTOR signaling pathway and activation of macrophages. TSF deserves much consideration as a potential therapeutic agent for NAFLD. |
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